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31 Springael, D. et al. (2002) Community shifts in a seeded 3-chlor-obenzoate degrading membrane biofilm reactor: indications forinvolvement of in situ horizontal transfer of the clc-element frominoculum to contaminant bacteria. Environ. Microbiol. 4, 70–80

32 Sentchillo, V. et al. (2003) Unusual integrase gene expression onthe clc genomic island in Pseudomonas sp. B13. J. Bacteriol. 185,4530–4538

33 Molin, S. and Nielsen, T.T. (2003) Gene transfer occurs with enhancedefficiency in biofilms and induces stabilization of the biofilm structure.Curr. Opin. Biotechnol. 14, 255–261

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0966-842X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tim.2003.12.010

The upper temperature for life – where do we drawthe line?

D.A. Cowan

Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa

A new archaeal isolate has been reported that is

capable of growing at up to 121 8C. This hyperthermo-

phile, dubbed strain 121, grows chemoautotrophically

using formate as an electron donor and FeIII as an elec-

tron acceptor and is closely related to members of the

archaeal genera Pyrodictium and Pyrobaculum.

Although the reported maximum growth temperature

of strain 121 is 8 8C higher than the previous record

holder (Pyrolobus fumarii; Tmax 5 113 8C), the two

organisms have virtually the same optimal growth

temperatures.

A publication by Kashefi and Lovley [1] has once morebrought the issue of the upper temperature limit for life tothe attention of the scientific community. The isolation of ahyperthermophilic archaeal strain that grows optimally at105–107 8C, and maximally at 121 8C is reported. At firstinspection, this organism would appear to have moved thebar up by some 8 8C, the previous record having been heldby Pyrolobus fumarii [2]. However, some caution must beadvised in interpreting these results. Strain 121 isreported to have a doubling time of ,7 hours at 115 8Cand 24 hours at 121 8C. By contrast, P. fumarii showed nogrowth at 115 8C [2], but had a doubling time of ,8 hoursat 112 8C with a significant number of cells in anexponentially growing culture surviving autoclaving.Therefore, strain 121 shows detectable growth at 8 8Cabove that reported for P. fumarii. Readers should notethat measuring accurate growth rates at extremes oftemperature is difficult at the best of times, and cannot bemade easier by the generation of the amorphous, opaquemagnetite that is the product of FeII reduction. However, itis remarkable that strain 121 survives at 130 8C for shortperiods. To put this in perspective, if 107 cells were exposedto 130 8C and completely killed in 2 hours, we would

calculate a survival half-life of ,6 minutes. The remark-able fact is that a vegetative cell can survive this long atsuch temperatures.

This work therefore represents another contribution tothe exciting search for the upper temperature limit for life:a search that has shown a generally linear rise (Figure 1)over the past 30 years.

Although the authors note that growth at 121 8C isremarkable because this is the temperature of steriliza-tion, there is nothing remarkable about this temperature

Figure 1. The rise of the upper temperature limit of life. Data points indicate the

dates that new organisms were reported that have significantly extended the

upper temperature limit of life. Included are Bacillus stearothermophilus [23],

Thermus aquaticus [3], Sulfolobus acidocaldarius [24], Thermoproteus tenax [25],

Pyrodictium occultum [26], Pyrolobus fumarii [2] and Strain 121 [1].

TRENDS in Microbiology

Sulfolobus acidocaldarius

Thermoproteus tenax

Pyrodictium occultum

Thermus aquaticus

Pyrolobus fumarii

Bacillus stearothermophilus

Strain121

Max

imum

gro

wth

tem

pera

ture

(°C

)

50

60

130

70

90

80

110

100

120

<1960 1970 1980 1990 2000 2010

Corresponding author: D.A. Cowan (dcowan@uwc.ac.za).

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per se other than the fact that this is the boilingtemperature of water at 1 atmosphere (15 psi) over-pressure. Had the original designers of autoclaves chosena different overpressure (such as 20 psi), a different‘sterilization’ temperature would have been embedded inour minds.

It is an exciting thought that the cardinal growthtemperatures determined in laboratory studies might beunderestimates of true in vivo growth temperatures. Forexample, the well-known extremely thermophilic aerobeThermus aquaticus [3] is readily isolated from terrestrial90–100 8C hydrothermal systems, but will not growreliably in the laboratory above 80 8C (H.W. Morgan,pers. commun.). Nevertheless, terrestrial and shallowmarine environments are probably too cool to harbor themost hyperthermophilic organisms, because their slowgrowth rates at such relatively low temperatures probablyresult in their being out-competed by rapidly growingorganisms, such as Pyrococcus [4].

The deep-sea hydrothermal vent biotopes, from whichnumerous hyperthermophilic archaea have been isolated,provide ample scope for high temperature growth. Thefriable metal sulfide walls of ‘smoker’ chimneys offer huge,if rather steep temperature gradients. The temperature ofthe interior water column might be as high as 400 8C,whereas the external seawater temperature is typicallyless than 5 8C, generating a temperature gradient ofseveral hundred degrees over a few tens of centimeters inthe chimney walls [5]. Horizontal transect samples fromsmoker chimneys have shown that biological signals, inthe form of amplifiable 16S rRNA genes, are present acrossmuch of the transect [5,6]. Unfortunately, the fragility ofthe chimneys and the difficulties of maneuvering instru-ments using the remote arms of submersibles mean that ithas not been possible to correlate the biological signatureswith the thermal gradient. To do so would provide anelegant means of providing a culture-independentmeasure of the upper temperature limit for life.

The molecular basis of thermophily

The enthusiastic analysis of molecular stability [7–10],which has closely followed the growth of the field of‘thermophily’, appears to have waned in the past fiveyears, leaving many fundamental questions unanswered.It would therefore be beneficial for thermophile research ingeneral if the Kashefi and Lovley publication [1] triggeredanother debate on the molecular mechanisms that dictatethe ability (or inability) of an organism to grow at hightemperatures. Protein structural analysis [7,10] hasindicated that conformational stability might not be thelimiting factor; proteins possessing high levels ofthermostability (Tm values in excess of 130 8C [11]) havebeen characterized. Strain 121 will probably yield moreexamples of thermostable proteins, the study of whichshould further contribute to our understanding of themolecular basis of protein stability. For example, a high-resolution crystal structure of the glutamate dehydrogen-ase will probably be a very useful addition to the proteinseries that exposed the vital contribution of salt-bridgenetworks to protein conformational stability [12,13].

Although we believe that we have at least some

understanding of protein structural stability, our under-standing of the implications of chemical instability at hightemperatures is less developed. Studies from the early1980s indicated that natural amino acids are reasonablychemically stable up to around 150 8C, but are degraded inminutes or even seconds at 250 8C [10,14]. Therefore, theconformational stabilty of proteins and the inherentchemical stability of their constituent amino acidssuggests that hyperthermophiles should not face aparticularly high maintenance energy burden owing toan increased protein for protein structural repair orresynthesis [10]. The same might not apply to other cellconstituents, such as coenzymes [e.g. NAD/P(H)] and lowmolecular weight metabolites (e.g. AMP, ADP, ATP, acetylCoA, acetyl phosphate and carbamoyl phosphate).

It has been suggested that small molecules, notmacromolecules, dictate the upper temperature limit oflife. This view is based on observations (albeit in vitro) thatkey metabolic intermediates, such as the nicotinamidecofactors, are highly unstable at temperatures as low as95 8C [15]. Strain 121 should provide an excellent test-bedfor analysis of putative mechanisms of cofactor stabiliz-ation (such as molecular channeling [16]) or of anypropensity to rely on more stable alternative chemicalmoieties (such as Fe-S proteins instead of nicotinamidecofactors [17]).

Origins of life

Kashefi and Lovley [1] do not report where the 16S rRNAsequence is positioned in the universal tree of life [18].However, its high relatedness to Pyrodictium and Pyro-baculum appears to position it as a relatively deep branchwithin the Crenarchaeotal lineage. This branching point,together with the apparently ‘primitive’ form [19] ofchemoautotrophic energy transfer (formate–FIII redoxpair) in strain 121, is consistent with the currently favoredview [20,21] of a high temperature – rather than lowtemperature – origin of life (or at least a thermophilic LastCommon Ancestor).

To sum up

The newly isolated hyperthermophilic archaeal strain 121grows slowly at 121 8C and even survives short periods at130 8C. This is another organism that grows best attemperatures well in excess of 100 8C! We should not beastonished so much by the numerical increments but bythe biochemical implications of this fact, and we should beexcited by the scope provided by this and similarorganisms to further our understanding of the evolutionand adaptation of molecular structures and systems.

And what about the upper limit of life? It appearsimprobable that the end-point of this search is representedby strain 121. The consensus view is that the true upperlimit, where the energetic burden imposed by molecularrepair and resynthesis becomes unsustainable, willprobably lie in the region of 140–150 8C [22].

References

1 Kashefi, K. and Lovley, D. (2003) Extending the upper temperaturelimit for life. Science 301, 934

2 Blochl, E. et al. (1997) Pyrolobus fumarii, gen. and sp. nov., represents

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a novel group of archaea, extending the upper temperature limit of lifeto 113 8C. Extremophiles 1, 14–21

3 Brock, T.D. and Freeze, H. (1969) Thermus aquaticus gen. n. and sp. n.a non-sporulating extreme thermophile. J. Bacteriol. 98, 289–297

4 Fiala, G. and Stetter, K.O. (1986) Pyrococcus furiosus sp. nov.represents a novel genus of marine heterotrophic archaeabacteriagrowing optimally at 100 8C. Arch. Microbiol. 145, 56–61

5 Takai, K. et al. (2001) Distribution of archaea in a black smokerchimney structure. Appl. Environ. Microbiol. 67, 3618–3629

6 Schrenk, M.O. et al. (2003) Incidence and diversity of microorganismswithin the walls of an active deep-sea sulfide chimney. Appl. Environ.Microbiol. 69, 3580–3592

7 Robb, F.T. and Clark, D.S. (1999) Adaptation of proteins fromhyperthermophiles to high pressure and high temperature. J. Mol.Microbiol. Biotechnol. 1, 101–105

8 Daniel, R.M. and Cowan, D.A. (2000) Biomolecular stability and life athigh temperatures. Cell. Mol. Life Sci. 57, 250–264

9 Daniel, R.M. et al. (1996) The denaturation and degradation of stableenzymes at high temperatures. Biochem. J. 317, 1–11

10 Jaenicke, R. et al. (1996) Structure and stability of hyperstableproteins. Adv. Protein Chem. 48, 181–269

11 Adams, M.W.W. (1993) Enzymes and proteins from organisms thatgrow near and above 100 degrees C. Annu. Rev. Microbiol. 47, 627–658

12 Britton, K.L. et al. (1999) Structure determination of the glutamatedehydrogenase from the hyperthermophile Thermococcus litoralis andits comparison with that from Pyrococcus furiosus. J. Mol. Biol. 293,1121–1132

13 Vetriani, C. et al. (1998) Protein thermostability above 100 degrees C:a key role for ionic interactions. Proc. Natl. Acad. Sci. U. S. A. 95,12300–12305

14 White, R.H. (1984) Hydrolytic stability of biomolecules at hightemperatures and its implication for life at 250 degrees C. Nature310, 430–432

15 Hudson, R.C. et al. (1993) Glutamate dehydrogenase from theextremely thermophilic archaebacterial isolate AN1. Biochim. Bio-phys. Acta 1202, 244–250

16 Van der Casteele, M. et al. (1990) Pathways of arginine biosynthesis inextremely thermophilic archaeo- and eu-bacteria. J. Gen. Microbiol.

136, 1177–118317 Daniel, R.M. and Danson, M.J. (1995) Did primitive microorganisms

use non-heme iron proteins in place of NAD/P? J. Mol. Evol. 40,559–563

18 Pace, N.R. (1997) A molecular view of microbial diversity and thebiosphere. Science 276, 734–740

19 Vargas, M. et al. (1998) Microbiological evidence for Fe(III) reductionon early Earth. Nature 395, 65–67

20 Wachtershouser, G. (1998) A case for a hyperthermophilicchemolithoautotrophic origin of life in an iron-sulfur world. InThermophiles: The Keys to Molecular Evolution and the Origins ofLife? (Wiegel, J. and Adams, M.W.W., eds), pp. 47–58, Taylor andFrancis

21 Di Giulio, M. (2003) The ancestor of the Bacteria domain was ahyperthermophile. J. Theor. Biol. 224, 277–283

22 Stetter, K.O. (1998) Hyperthermophiles: Isolation classification andproperties. In Extremophiles: Microbial life in Extreme Environments(Horikoshi, K. and Grant, W.D., eds), pp. 1–24, Wiley-Liss

23 Sharp, R.J. et al. (1991) Heterotrophic thermophilic Bacilli. InThermophilic Bacteria (Kristjansson, J., ed.), pp. 19–50, CRCPress

24 Brock, T.D. et al. (1972) Sulfolobus: a new genus of sulfur oxidizingbacteria living at low pH and high temperature. Arch. Mikrobiol. 84,54–68

25 Zillig, W. et al. (1981) Thermoproteales: a novel type of extremelythermoacidophilic anaerobic archaebacteria isolated from Icelandicsolfataras. Zbl. Bakt. Hyg. I. Abt. Orig. C2, 205–227

26 Stetter, K.O. et al. (1983) Pyrodictium gen. nov., a new genus ofsubmarine disc-shaped sulfur reducing archaebacteria growingoptimally at 105 8C. Syst. Appl. Microbiol. 4, 535–551

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|Research Focus Response

Response to Cowan: The upper temperature for life –where do we draw the line?

Kazem Kashefi

Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA

It was Louis Pasteur in the 19th century who made theconnection between spoiled food and microorganisms anddiscovered that heat treatment could be used to kill themicrobes that caused certain products to spoil. A series ofexperiments led the French microbiologist to determinethat temperatures in the order of 1108C destroyed allbacteria, therefore negating the popular theory of spon-taneous generation [1]. Pasteur also tested the heatresistance of fungal spores in a dry oven and found thata one-hour sterilization at 120–1258C was enough to killthe recalcitrant spores [1]. Sterilization standards were,therefore, not the arbitrary caprice of an impulsive mindbut the careful scientific product of one of the best inqui-sitive scientific minds in microbiology. In today’s auto-claves, a combination of pressure and steam to reachtemperatures in the order of 1218C has achieved what

Pasteur did more than a 100 years ago: to kill even themost resistant forms of life known to us.

Little could Pasteur predict that, more than a centurylater, a microorganism known as strain 121 would beisolated from a hydrothermal vent that could not only growat the standard 1218C temperatures of modern-day auto-claves, but could survive temperatures as high as 1308C[2], therefore raising all known standards for the highesttemperature for growth and survival of any living being.As stated in the article describing this discovery, ‘Theupper temperature limit for life is a key parameter fordelimiting when and where life might have evolved on ahot, early Earth; the depth to which life exists in theEarth’s subsurface; and the potential for life in hot,extraterrestrial environments.’ [2].

By growing at autoclave temperatures, strain 121raised the known upper temperature for growth of anyliving organism 88C higher than the previously recognizedCorresponding author: Kazem Kashefi (kkashefi@microbio.umass.edu).

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